CZ38194U1 - A fungicide - Google Patents

Description

Fungicidal preparation

Field of technology

The field of technology relates to a biological contact broad-spectrum fungicidal preparation (III) pre- and post-infectious fungicide (pre-harvest for plants and post-harvest for so-called fumigation), with fungicidal, virucidal and bactericidal effects. At the same time, it has biodegradability and good biocompatibility, while natural biofungicides can be cheaper and more available than synthetic fungicides and also have no side effects unlike synthetic fungicides.

We focus on the area of bio-substances: Acetic acid (ethanoic) 10%, active substance, organic acid.

Other substances with fungicidal and antibacterial effects are - L-ascorbic acid (the so-called phytovaccine), since numerous studies have shown that it has a wide range of antimicrobial activities and acts as a botanical fungicide that inhibits the germination of spores and the growth of plant pathogens.

This is followed by SA - Salicylic acid (so-called phytohormone), which in plants plays a fundamental role in the activation of plant resistance systems to diseases, including immunity triggered by a pattern - effector (a small molecule that selectively binds to a protein and regulates its biological activity) and systemically acquired resistance. It is a phytohormone that inhibits FoTOR complex 1 (FoTORCl) by activating FoSNFl in vivo, e.g. transgenic potato plants with interference of FoTOR1 and FoSAH1 genes inhibited the invasive growth of hyphae (fungal filaments) and significantly prevented the occurrence of Fusarium wilt. - activates the immune system of plants. SA can effectively stop hyphal growth, spore production and pathogenicity, eg Fusarium wilt.

The preparation also contains another natural substance - Glycerin, natural adjuvant, synergist, surfactant.

It is a technology - based on a biofungicide, aimed at the treatment of foliar diseases, including the so-called post-harvest application (fumigation) to crops. It is a composition - concentrate, for subsequent dilution of the product before use, which is intended as a fungicidal product for plants, fumigation of plants, etc.

Bio-fungicide can be used as a safe alternative in organic farming. The composition can be used, for example, in the treatment of fungi on crops, vegetables and other plants.

The preparation also includes the area of so-called crop fumigation (fumigation/disinsection of fruits, grains, etc.), it is the so-called post-harvest treatment of crops before large-capacity storage.

Current state of the art

The intensive use of synthetic fungicides causes serious environmental and public health problems. It is therefore advisable to develop new fungicidal preparations that are effective and at the same time safer for workers and the environment, while they should include a low price/production ratio. In this sense, organic fungicide formulations have become necessary in recent years.

A fungicide is a form of pesticide that is used to protect plants (or crops) from certain types of fungi and other diseases that attack plants. Its role is to inhibit the given unwanted organism in the attacked plant. To control the occurrence and action of pathogens

- 1 CZ 38194 U1 in stands, not only for agricultural crops, it is currently advisable to choose effective protective agents, with increasing emphasis on minimizing the use of chemical agents.

The use of synthetic chemical compounds can have a positive effect on crop yields, but it can also result in secondary effects on treated plants, on shelf life affecting the appearance and especially the nutritional value of crops based on unexpected effects, on susceptibility to browning by influencing phenolic compounds and enzymes, etc. .

At the same time, negative effects of synthetic substances can occur when consuming crops treated with these substances. Therefore, biological methods of protection are increasingly sought after, which focus on the detection and elimination of pathogens (organisms capable of causing disease) affecting plants, e.g. yellowing of leaves, spots on leaves, stunted growth, rot, wilting, curling of leaves, etc.

Plant pathogens include disease-causing agents, especially fungi (fungi), as well as viruses, viroids, phytoplasmas and bacteria:

• fungal disease - elimination using a fungicidal spray designed for plants • bacterial infection - elimination using bactericidal sprays that are safe even for young plants (e.g. used by us - Ethanoic acid, Salicylic acid) • viral disease - elimination using components (e.g. acid Salicylic and ascorbic acids)

E.g. grapevines are most often attacked by fungal diseases, which include Downy mildew (Peronospora), Downy mildew (Moučenka), Gray mold (Botrytida).

In addition to timely intervention with suitable fungicides, prevention is also essential, which starts with planting bushes in a suitable location, continues with balanced fertilization and illumination of the bushes. Viral diseases transmitted by sucking insects are also feared.

Specific fungi attacking agricultural products:

Grape downy mildew (perenospora) - yellowish spots or small demarcated spots appear on the leaves around the nervature (system of vessels, veins in plant leaves). On the underside, these spots are covered with a whitish coating of the fungus. The spots gradually dry up and severely affected leaves fall off. Attacked flowers and parts or whole young grapes also dry out. Berries are directly attacked only up to a size of 2.5 mm. Damaged berries are deformed and turn gray-green. A stronger attack on the leaves results in the non-ripening of the shoots and thus an increased risk of frost damage.

The cause of grape downy mildew is the microscopic fungus Plasmopara viticola, which mainly overwinters in infected leaves, which are the primary source of spread in the spring. Since it is necessary to moisten the leaves for infection, the disease is promoted by warmer and above all rainy weather. As a preventive measure, fungicide spraying is applied to these diseases immediately after flowering and then approx. in a week. If the weather is extremely favorable for the development of the disease, the vine can be treated with a fungicide even before flowering. If the warm and humid weather continues in July, you can continue the treatment with fungicides.

Botrytis cinerea (gray mold) - an important postharvest pathogen for grapes. Identifying gray mold is not difficult. It produces an initially white to gray, later gray-brown coating, surface or intramatrical hyphae that are hyaline (translucent) to brown.

Vine powdery mildew (oidium, powdery mildew) - forms on shoots, leaves, inflorescences and grapes where parasitic fungi form whitish powdery coatings. The affected surface tissues die, turn gray and their deformations occur. Inflorescences are showered and young berries dry up, crack and their protrusion occurs (seed perforation or "seed protrusion" - extrusion). The disease is also referred to as oidium or "powdery mildew". The fungus overwinters mainly in its form

- 2 CZ 38194 U1 undergrowth (mycelia) in buds or in the form of fruiting bodies. The development of the disease is supported by alternating air humidity (dews, fogs, showers), temperatures of 24 to 30 °C, an excess of nitrogen, a lack of potassium and dense plantings. The most intensive spread usually occurs during June. The more susceptible varieties include, for example - Aurelius, Frankovka, Irsay Oliver, Blue Portugal, Muller Turgau, Neuburg, Pálava, Riesling Vlasský, Grüner Sylvania and Grüner Veltliner.

Gray mold (Botrytis cinerea) - on vines. The first symptoms of gray mold usually appear on the grapes and leaves. A gray, wavy mold forms on the surface of the grapes, which can eventually cover the entire grape. The disease can also affect the flowers, which can rot and fall off. The disease causes the greatest damage by attacking ripening and ripe grapes, on the berries of which darker spots are visible and later the skin cracks and peels.

Infested berries are covered with a gray coating of conidiophores. In suitable weather, the infected berries mummify. The attack itself can occur during several stages. First, it is during flowering, when unfallen flower caps can serve as a place for infection. Another period that is most suitable for mold is when the grapes are closed and the subsequent development, ripening and ripening of the fruits.

Other grapevine pathogens:

Grapevine Gray Rot (Grey Mold), Grapevine Leaf Roll, Grapevine Roncent (Roncent Virus), Red Blight, Wood Diseases, White Rot of Grapevine, Bacterial Diseases, Phytoplasmic Yellowing and Reddening of Grapevine Leaves (Strolbur).

Year-round application of fungicides to hops is the primary method for managing hop blight in susceptible and some moderately resistant cultivars (variety or variety).

Humulus lupulus - spinning hop:

Peronospora chmelová (hop mold) - as the main fungal disease, it attacks already emerging shoots, later the leaves and then the heads. The infestation is more pronounced in wet and warm seasons. Yellow-green spots appear on the leaves, which later turn brown and die, the assimilation area of the leaves is reduced with fewer flowers. The bracts of the heads turn brown (rusty tinge), the heads have worse signs and properties assessed during commercial evaluation.

We provide protection against Peronospora in the form of 4 to 5 protective interventions in the period from approximately mid-June to mid-August. For this, so-called treatment signaling and short-term occurrence forecasts based on daily monitoring of temperature, relative air humidity and total precipitation are used. In extremely wet years and with insufficient protection, Peronospora can cause serious economic damage.

Powdery mildew of hops - a disease belonging to the group of mycoses, was practically a less significant disease in the past and protection against it was not routinely carried out. A more pronounced onset of its expansion occurs from the mid-1990s (greater sensitivity in meristem stands). Smaller white spots are formed on the upper side of the leaves, which turn into powdery coatings, the infection and the whitish coating gradually moves to the heads as well.

Infested heads smell unpleasant and adversely affect the taste and aroma of beer. In the case of favorable conditions for the development of the disease (drier weather), it is necessary to carry out two preventive chemical treatments (before flowering, after the beginning of flowering). Fallen hops can also attack sprouting shoots in spring.

Hop curliness - manifests itself in the yellowing and twisting of the vine leaves, the leaves are fragile and break easily. In our conditions, the symptoms are mainly caused by a lack of zinc in the plants in June and July.

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Hop aphid - this harms by sucking plant juices on the underside of the leaves and leaves sticky sweet excrement (honeydew) on them, which is also a breeding ground for the spread of saprophytic fungi (black). After the heads are attacked in a similar way, the yield and especially their quality decreases.

Whitefly - by sucking on the leaves, it causes blisters, the leaves turn yellow and turn gray, with a strong occurrence they dry up and fall off. If timely protective measures are not taken, the svilushka also passes into the heads.

Hop beetle and phytophagous bugs - in the spring they attack sprouting and young shoots, later young leaves. Both are considered so-called minor pests and chemical intervention is only carried out in case of a stronger occurrence.

Verticillium wilt of hops - the speed and severity of the disease is inversely proportional to the temperature of the soil. This hop wilting is promoted by mild temperatures and suppressed by high temperatures. The disease can have a worse course in the presence of phytopathogenic nematodes, and its development is supported by the use of large doses of nitrogen fertilizers.

Basal cortical necrosis of hops - Fusariosis (wilt). Most often the species Fusarium sambucinum, then Fusarium avenaceum. This infection manifests itself differently depending on which part of the plant is attacked. The base of the vine tends to be swollen, as the new wood thickens, the underground part of the vine is usually the most strongly attacked, the mycelium of the pathogen grows through the vascular bundles of the hops and roots, blocking the supply of water and nutrients.

This leads to rapid wilting of the above-ground part. Whitish coatings of mycelium or whitish clumps - sporodochia - appear on the bark of the elderberry. This leads to rapid wilting of the above-ground part.

Some of the important soil pathogens are - Fusarium, Rhizoctonia, Macrophomina and Sclerotinia

Overview of the most common plant diseases:

• The cause of neck rot - Fusarium wilt, is a fungal disease of plants caused by fungi of the sickle genus (Fusarium) • Gray mold - Botrytis cinerea • Powdery mildew - Blumeria, Erysiphe • Grapevine powdery mildew - Uncinula necator • Grape mold - Plasmopara viticola • Fruit rot - moniliosis ( apples, pears, etc.) Monilinia fructigena • Disease of above-ground parts of trees - Phytophtora (or root disease) • Downy mildew on wheat, corn - Ustilago • Causes of rust - Melampsora, Puccinia, Uromyces, a group of parasitic peduncle fungi, these are plant parasites, which form the undergrowth or mycelium • Root and stem rot - Sclerotinia sclerotiorum • Bacterial rot, bacterial wilt, bacterial leaf spot

In general about diseases related to groups of plants:

Powdery mildew (Erysiphales) - is the most common fungal disease that attacks both useful and ornamental plants, and is characterized by a white coating that forms mainly on the leaves of plants (e.g. powdery mildew of vines, powdery mildew of potatoes, rapeseed, hops, pumpkins, powdery mildew of peppers, courgettes, cucumbers, cruciferous vegetables, peas, hops, apples, clovers, strawberries, canola powdery mildew, true powdery mildew, flax powdery mildew, beetroot, poppies, roses, ornamental plants, etc.).

Powdery mildew (Erysiphe cruciferarum) - is a white cobweb that later turns into a powdery coating. Powdery mildew is most often found on roses, perennials, vegetables and fruit bushes.

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Nectria galligena - is a fungal disease on the branches of shrubs or trees caused by the fungus Nectria galligena, the origin of which is the ruddy rose, which mainly attacks currants, hawthorns, lindens, beeches and gooseberries.

Potato blight (caused by the fungus Phytophthora infestans) - attacking not only potatoes but also tomatoes and other eggplant-like plants is a mold that is literally a disaster.

Cucumber blight (Pseudoperonospora cubensis) - in addition to all types and types of cucumbers, it also attacks candied melons, showing yellow spots on the leaves.

Cruciferous mildew (Hyaloperonospora parasitica) - you can recognize it by the yellow-brown or yellowish spots that form on the outer leaves - lettuce, cabbage, cauliflower, kohlrabi, cabbage, broccoli, radish, watercress, horseradish, etc., which later turn brown.

Tomato blight - the fungus that causes tomato blight, and it is a very resistant type of fungus.

Scab of stone fruits, Moniliosis (Venturia carpophila) - is the most common fungal disease of apple, pear, apricot, but also peach, cherry and apricot (a fungal disease that damages leaves, fruits and flowers). Scab and powdery mildew of apples and pears (pear rust) are also among the most common diseases.

The fruit weevil is one of the 140 species of weevils, which mainly parasitizes fruit trees. Especially on apple, pear, plum and grape trees. Adults grow up to approx. 0.5 mm, are colored red.

Root neck rot (Phytophthora) - a disease caused by fungi is quite common and can affect various types of plants, including some types of vegetables.

Leaf curl (Taphrina deformans) - these are twisted and curled leaves with prominent red raised blisters, they do not only affect peaches, they can also affect nectarines.

Black spot of roses (Diplocarpon rosae) - which attacks the leaves of roses where you notice black spots, while they are attacked by a fungal disease.

Lawn diseases - fusarium spots, corticid disease, snow mold occurring after the winter period, etc.

Classification of fungicides from a chemical point of view:

- biological fungicide with minimization of residues

- systemic fungicide spray protects the entire plant, therefore the entire plant does not have to be perfectly covered during application, the active substance is distributed throughout the plant

- sulfur fungicides

- copper compounds

- mercury compounds

- compounds of other heavy metals

Mycelium: or hyphae - a tangle of fibers is also referred to as mycelium, which is a cluster of interwoven fibers, characteristic especially of fungi and some bacteria. The fibers can be divided by septa (partitions) into individual cells, or these septa are absent and the entire mycelium is made up of one cell.

Mycelium penetrating the soil is called basal (vegetative) mycelium, the part above the soil is aerial or reproductive mycelium (if it forms spores).

• primary mycelium - one nuclear (monokaryotic), i.e. there is only one nucleus in each cell

- 5 CZ 38194 U1 • secondary mycelium - binucleate (dikaryotic), i.e. there are two nuclei in each cell • tertiary mycelium - also binucleate, but it creates specialized tissues of fruiting bodies, such as pseudoparenchyma (fungal false tissue, which is indeed composed of hyphae, but these have lost their fibrous character and become crowded into a compact structure, or plectenchyma (false sponge tissue)

Bio-fungicides are a new approach to combating plant diseases. This means that these sprays against fungi, rots, etc., are biological fungicides used to kill fungi, etc., which attack plants/fruits and cause economic damage to them. The chemical molecules used as fungicides are formulated in a combination of different substances.

Formulation and methods of application using bio-fungicides: spraying plants and soil, fumigation

- the application is applied by spraying the leaf/plant with a fungicide

- one of the most effective uses of biofungicides is - as a preventive treatment in growing substrates, i.e. material for growing plants, i.e. it is a soil spray

- fumigation (wet treatment of crops and seeds) - post-harvest treatment before storage - seeds, fruits, vegetables (treatment of crops and seeds after harvest), fumigation effect - means that the active substance starts to evaporate on the harvested crop, thereby reaching the mycelium (execution in fumigation tubs, steam fumigation of grains, etc.)

This means that Bio-fungicide is a product suitable for fumigation of seeds and crops (fumigation / disinsection of cereal grains, fruit, etc.), it is a post-harvest treatment of crops before storage, i.e., in warehouses, silos, etc. So-called, post-harvest the application of fungicides (fumigation) is used in order to reduce losses (based on attacks by various types of fungi) on stored crops.

Using multi-component bio-technology, we formulate chemical molecules used as fungicides in a combination of different substances, aiming to maximize the biofungicide application and its effectiveness, with sustainable agricultural practices and environmental friendliness. This means that the concentration in the environment has to be very high initially, then quickly drops to a low and ineffective level during application. In contrast, bio-fungicides with a specialized surfactant/adjuvant show lower initial concentrations of the active ingredient that remain stable long enough on the leaf.

Biofungicides are living organisms (more specifically microorganisms) or products derived from microorganisms, including natural metabolites produced by these organisms during their growth and development. Limitations for the development of biofungicides, especially those containing biotic agents (a component of nature that is represented by organisms together with their mutual relationships), include specific requirements for maintaining the activity and effectiveness of the organism, which requires the development of special preparations.

Biofungicides, also known as natural fungicides, play a key role in sustainable agriculture by effectively inhibiting the development and growth of pathogens on plants and in the soil. At the same time, they help increase crop productivity by reducing competition between plants for nutrients, water and light. Their ecological nature distinguishes biofungicides from synthetic substances and supports ecological agricultural practices. Despite their numerous advantages, many biofungicidal compounds are inherently unstable and prone to degradation in environments with higher temperature, light, moisture and microbial activity, which poses a challenge for new effective fungicide formulations.

To address this problem of instability, new biotechnology strategies have been increasingly emphasized in recent years, offering the following advantages for biofungicides:

• reduction of the amount of chemical (synthetic) substances needed for growing crops • improved effectiveness of the product as a whole • sustainable (gradual) release of biological compounds

- 6 CZ 38194 U1 • use in eco-agricultural and natural systems • protection of environmental factors • potential of improved formulation systems to help solve plant pathogen problems

Especially with the use of new substances, they are promising materials for bio fungicides. These new substances are specially designed for the targeted delivery of biofungicides, ensuring effective crop protection. The inclusion of new substances in the development of these preparations revealed their remarkable potential. Using new technologies, researchers aim to maximize the effect of biofungicide applications and their effectiveness, to provide potential sustainable solutions for plant pathogen control.

An emerging research area that has not been applied to biological plant protection, while having the potential for innovative developments that could be achieved through variations in fungicide and synergistic technologies. The aim is to develop an understanding of the factors influencing the formulation of bio-fungicides for successful use in agricultural events. natural ecosystems and assess the prospects for the development of new technological solutions for improved formulation of biofungicides. The potential of improved systems of these formulations may help solve current plant pathogen problems, with commercial products likely to emerge that combine the benefits of biofungicides.

According to research to date, only a portion of common fungicides are absorbed by plants, the rest being lost in one or more of the following ways:

• evaporation, adsorption (accumulation of liquid particles) • leaching • photodecomposition (absorption of radiation leading to the formation of molecular fragments) • chemical degradation and microbial decomposition

The continuous use of synthetic fungicides leads to the development of resistance of pathogens to synthetic fungicides. The science of biofungicide technology can be used as a tool to produce a slower release fungicide (eg Glycerin) to achieve conditions throughout the plant growing season without disrupting the environment. Studies have shown that bio fungicides can produce more targeted and less toxic formulations for agricultural applications. The new formulations will allow the application of lower doses of fungicides, which is desirable because we are reducing the long-term effects of fungicide residues in agricultural areas and their toxicity to the environment. These natural preparations can facilitate the easy delivery of biofungicides to plants and reduce residual accumulation in the soil.

Modification of the surface or volume properties of fungicides has great potential for effective improvement of agricultural productivity, e.g. they strengthen tolerance mechanisms in plants under stressful conditions (attack by pathogens). In this regard, these new bio fungicides can be considered as effective tools to overcome some of the main challenges of sustainable agricultural production.

At the same time, other findings are used, e.g. - in 2013, the European Commission approved vitamin C as an effective substance for the treatment of potatoes and tomatoes against plant pathogens such as: Phytophthora infestans, Botrytis sp. and Fusarium sp.

Treatment of foliar diseases on vegetables with ascorbic acid was able to significantly reduce powdery mildew infestation (AbdelKader et al., 2012; Abdel-Kader & El-Mougy, 2014).

Other studies show that vitamin C can show significant fungicidal activity against Fusarium graminearum, Alternaria alternata and Pyrenophora tritici-repentis under in vitro conditions (Shomeet et al., 2018) and also against Magnaporthiopsis maydis, which causes maize late wilt disease (Abdel-Kader et al. al., 2022).

Other research confirms that ascorbic acid can reduce linear growth on plant pathogens,

- 7 CZ 38194 UI such as Fusarium oxysporum, F. solani and Macrophomina phaseolina (Hassan et koi., 2014).

In his research, Christian Chervin confirms that several small molecules of natural origin such as acetaldehyde, acetic acid, ascorbic acid, ethanol, ethylene, jasmonic acid and methyl jasmonate, salicylic acid and methyl salicylate and est. have been shown to act as alternatives to synthetic fungicides for biological protection against fungal diseases of grapes and other crops (Chervin, 2012).

Article 1

Microbial biofungicides as a substitute for chemical fungicides in phytopathogen control: current perspectives and research directions Hindawi Scientific Year 2024, Article ID 5322696, 12 pageshttps://doi.org/10.1155/2024/5322696

Lamenew Fenta, Habtamu Mekonnen

Department of Biology, Debre Markos University, Debre Markos, Ethiopia

Department of Biology, Bahir Dar University, Bahir Dar, Ethiopia

First published: 28 February 2024 Source https://doi.Org/10.l 155/2024/5322696

Source: https://onlinelibrary.wiley.com/doi/10.1155/2024/5322696

Sustainable agriculture is essential to preserve farmers' livelihoods, increase food and nutrition security, and sustain long-term national growth [ 5 ]. Improving or maintaining the quality of the environment while simultaneously protecting natural resources is a prerequisite for sustainable development [ 6 ]. Thus, sustainable agriculture requires efficient management of agricultural resources to control pathogens and diseases to the extent that they do not negatively affect crops by disrupting the natural balance [ 7 ]. Synthetic fungicides have been used to control crop diseases and increase crop production for many years [ 8 ].

Although synthetic fungicides reduce crop losses, excessive use of synthetic fungicides has led to pathogenicity resistance, pathogen resurgence, and pathogen extinction. They are also harmful to aquatic life, soil biodiversity, humans and animals [ 9 ].

Typical effects of these fungicides include soil embrittlement, reduced soil respiration, and reduced activity of several soil microorganisms [ 10 ]. Synthetic fungicides reduce animal vitality, immunity, and animal reproductive efficiency [ 11 ]. Synthetic fungicides have a detrimental effect on plant growth by reducing the biological function of soil microbes in the production of specific plant growth promoting properties such as indole-3-acetic acid, nitrogen and siderophores [ 12 1.

Fungicide leaks can enter water bodies and cause pollution and destruction of aquatic life. Furthermore, bioaccumulation of fungicides in the aquatic environment is associated with the development of fatal diseases such as diabetes, rash, kidney disease, and cancer in both animals and humans as well as aquatic organisms [ 13 ]. The EU and the US have already outlawed some chemical pesticides due to their harmful effects, and many conventional items have also been phased out due to concerns about their effects on the environment and human health [ 14 ].

Limited biodegradability and high persistence of synthetic pesticides are some major disadvantages [ 15 ]. Contamination of the environment (water and soil), harmful effects of fungicide residues on beneficial insects (earthworms, bees and spiders) and harmful effects on soil microflora result in loss of biodiversity and disruption of nutrient cycling [ 16 ]. In these situations, an integrated strategy provides a range of management options that are sustainable and environmentally friendly while still protecting human health and the environment. Biological control, which uses active biological control agents to reduce pest damage, is a key component of a comprehensive Γ 17 1 strategy.

The primary idea is to use microbes and their products to suppress plant diseases without affecting the properties and elements of the ecological environment [ 18 ]. In the era of sustainable agriculture, microbial biofungicides provide solutions to problems including fungicide resistance, environmental problems, and human health problems Γ 19 1.

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Biofungicides are considered to be significantly more environmentally friendly than natural fungicides, yet this long-term option competes for attention in the current market with synthetic pathogens. Key issues related to technological difficulties and long-term sustainability require an urgent need for solutions for greater adaptability for the popularization or promotion of microbial biofUngicides. These microbial biofungicides can take the form of microbial fungicides (microbial origin) [ 20 ], phytofungicides (plant origin) [ 13 ] and nanobiofungicides (nanoparticles made from biological substances) [ 21 ].

Microbial biofungicides are cheaper, more available and more durable than synthetic fungicides and also have no adverse effects unlike synthetic fungicides [ 22 ].

Phytofungicides, in addition to containing diverse phytochemical components that give them different modes of action, are less hazardous to human health than synthetic fungicides [ 23 ]. Nanobiofungicides outperform synthetic fungicides in terms of fungicidal effect, controlled or targeted release, biodegradability, and good biocompatibility [ 21 ].

Therefore, the main objective of this review is to assess the progress of microbial fungicides, their potential to replace chemical fungicides, their disadvantages and to propose a basis for future research that will be most helpful in managing phytopathogens.

This review also discusses the effects of using synthetic fungicides to treat crop pathogens and explores the role of microbial biofungicides in plant disease management and outlines current trends and the status of utilization of these mechanisms.

Article 2

Profile of Citric Acid - An Active Ingredient Suitable for Minimal Risk Pesticide Use Brian P. Baker and Jennifer A. Grant

New York State Integrated Pest Management, Cornell University, Geneva NY

Source: https://ecommons.cornell.edu/server/api/core/bitstreams/3b5dfae4-953b-4591-b256-

81246b217385/content

Summary:

Citric acid is a naturally occurring substance and a common food ingredient. It is irritating and can cause other health problems in exposed persons. Its pesticidal use is primarily as an antimicrobial. EPA has confirmed that no additional data requirements need to be met on this basis, stating that citric acid can be used as an active ingredient in pesticides with minimal risk.

Pesticide use:

The main pesticidal use of citric acid is as a contact antimicrobial pesticide. It is used as a food contact and non-food contact antimicrobial agent with many uses in food handling, residential and public facilities.

Citric acid is used as a disinfectant for fruits, vegetables and food contact surfaces.

It is also used as a disinfectant, virucidal and germicide; and as an algicide in swimming pools, hot tubs and other places where unwanted algae grow.

Formulations and combinations:

As a pesticide active ingredient, it is often combined with acetic acid and malic acid. It is also combined with various essential oils, especially peppermint oils. Citric acid is an ingredient in registered antimicrobial pesticides and as an inert ingredient in a wide range of pesticides. It works

- 9 CZ 38194 U1 as a pH adjuster and chelating agent and as an adjuvant mixed with a wide range of pesticides as a pH adjuster and buffering agent in tank mixes.

General issues:

Citric acid is a Krebs cycle acid responsible for basic metabolic activity in all aerobic organisms and is closely related to malic, tartaric and fumaric acids.

Citric acid is a naturally occurring carboxylic acid found in fruits, vegetables and other plants. It is the predominant acid found in Citrus spp. fruits such as lemons, oranges and grapefruits. Tomatoes (Lycopersicon esculentum) also have a considerable amount.

Citric acid is also in fruit. Other plants known to contain high amounts of citric acid include angelica (Angelica archangelica), cranberries (Vaccinium macrocarpon), Gingko biloba, stinging nettle (Urtica dioica), rose hips (Rosa spp.), hibiscus (Hibiscus spp.) and marigolds (Tagetes spp.) (Khan and Abourashed 2010).

Originally isolated from lemon juice in 1784, commercial citric acid was produced by extraction from the juice until the 1940s.

Virtually all commercial citric acid is now produced by fermentation. Citric acid has a wide range of food and non-food uses that far exceed its use as a pesticide. The EPA first registered citric acid as a pesticide in the 1970s (US EPA OPPTS 1992).

Article 3

L-ascorbic acid: A multifunctional molecule supporting plant growth and development.

Daniel R. Gallie* Scientifica (Cairo).2013; 2013: 795964 PMCID: PMC3820358

Published online 2013 Jan 17. doi: 10.1155/2013/795964 PMID: 24278786

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820358/

Summary:

L-ascorbic acid/Asc (vitamin C) is as important to plants as it is to animals. Ascorbic acid functions as a major redox buffer and as a cofactor for enzymes involved in the regulation of photosynthesis, hormone biosynthesis and the regeneration of other antioxidants.

Ascorbic acid regulates cell division and growth and is involved in signal transmission. Unlike the single pathway responsible for ascorbic acid biosynthesis in animals, plants use multiple pathways to synthesize ascorbic acid, perhaps reflecting the importance of this molecule to plant health.

Due to the importance of ascorbic acid for human nutrition, several technologies have been developed to increase ascorbic acid content in plants through manipulation of biosynthetic or recycling pathways. This article provides an overview of these approaches as well as the consequences that changes in ascorbic acid content have on plant growth and function.

The ability of plants to tolerate changes in ascorbic acid content is discussed. However, the many functions that ascorbic acid fulfills in plants will require highly targeted approaches to improve their nutritional quality without compromising their health.

4.3. Ascorbic acid (Asc) regulates abiotic and biotic stress responses.

A reduction in Asc content may also affect resistance to pathogens. The vtc1 and vtc2 mutants are more resistant to Pseudomonas syringae and Peronospora parasitica infection, as the growth of the bacterial or fungal pathogen was substantially reduced [112].

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Greater resistance correlated with greater induction of pathogenesis-related proteins PR-1 and PR-5 after infection, as well as increased levels of salicylic acid [112], suggesting faster induction of defense responses when Asc levels are low.

10. Conclusions

In contrast to the single pathway responsible for Asc biosynthesis in animals, multiple Asc biosynthetic pathways are present in plants, possibly reflecting the importance of this molecule for plant health. The increase in atmospheric oxygen during Earth's history would have posed a particular challenge to terrestrial organisms, leading to a greater reliance on antioxidants to limit the harmful consequences of increased oxygen exposure.

All enzymes of the Smirnoff-Wheeler Asc biosynthetic pathway are present in algae [204], demonstrating the evolution of this pathway before the appearance of land plants. However, as an uncharged molecule that has a relatively long lifetime, H2O2 can freely pass through membranes, and thus diffusion from algae into the aqueous environment may provide another means of reducing their oxidative burden.

Since this pathway is not available to land plants, ascorbic acid (Asc) together with other antioxidants probably facilitated their colonization of soil.

From its role in the regulation of photosynthesis and as an antioxidant that detoxifies exogenously and endogenously generated ROS, through its role in the regulation of cell division and flowering, to its function as an enzyme cofactor in many enzymatic reactions, ascorbic acid has become essential for many aspects of plants.

Article 4 (from PDF)

Effect of acetic, formic and propionic acids on plant pathogenic fungi

Sercan Sehirli* and Cansu Saydam lUludag University, Faculty of Agriculture, Department of Plant Protection, 16059 Nilufer, Bursa, TURKEY

Received: 14/12/2016; Accepted: 23/12/2016; Published Online: 30.12.2016

Source: https://acikerisim.uludag.edu.tr/items/cc8c91b9-b073-422e-8ba7-c1a6af644b4b

Summary

The effectiveness of low or non-toxic chemicals is an alternative to the use of fungicides. In particular, GRAS - Generally Recognized as Safe compounds are quite suitable for preventing the development of plant diseases.

Propionic, formic, and acetic acids were selected to determine antifungal effects on some soil-borne plant pathogens that are listed as GRAS chemicals.

GRAS compounds were tested on Macrophomina phaseolina, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum and Rhizoctonia solani to understand the effectiveness of organic acids on the development of plant pathogens. Inhibition of mycelial growth by propionic, formic and acetic acids was determined. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the organic compounds were also determined.

Propionic acid was significantly stronger than formic and acetic acids. Propionic acid at a concentration of 0.7%, formic acid at a concentration of 0.9% and acetic acid at a concentration of 1.8% completely inhibited the growth of the mycelia of all fungi (mycelia - a cluster of intertwined fibers, characteristic especially of fungi and some bacteria). The effectiveness of organic compounds was variable and showed different effects on fungi based on their resistance. Resistance to, for example, B. cinerea, S. sclerotiorum and F. oxysporum was higher than that of R. solani and M. Phaseolina.

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INTRODUCTION

Simply put, GRAS (Generally Recognized as Safe) chemicals are environmentally friendly, low-toxic or non-toxic compounds that can come into contact with food and food additives (FDA 2016). Most GRAS chemicals are currently used in the food industry as food additives and consumed by humans. GRAS compounds are not only used in the food industry, but are also used in conventional agriculture, in agricultural pest control.

GRAS ingredients are allowed to be used in organic farming. One alternative is GRAS compounds - methods of synthetic pesticides to prevent plant pests (Corral et al. 1988).

Organic farming has strict rules and restrictions on the use of pesticides in relation to pesticide residues. In addition, synthetic pesticides are used in most countries but are not allowed in organic farming.

Thus, alternative methods or compounds are needed instead of using synthetic pesticides to prevent plant diseases. In addition, these pesticides pose a threat to human, animal and environmental health, as well as causing the development of resistance in plant pathogens.

The solution could be organic acids, against resistance problems and health risks caused by pesticides. The chemical bonds of organic acids and their structures are easily decomposed in the soil or on plant leaves. No harmful residues remain after degradation.

This is why these chemicals or compounds are environmentally friendly. For prevention, organic acids can be used for the development of plant diseases and reduce the mentioned risks.

In agriculture, soil-borne plant pathogens are one of the main problems (Koike et al. 2003). Soil-borne pathogens cause economic and yield losses in the field and even after harvest and during the storage period.

Soil-borne pathogens cause root decay, decline, softening of roots and plant death (Dreistadt S.H. 2001). Some of the important soil pathogens are Fusarium, Rhizoctonia, Macrophomina and Sclerotinia (Agrios 1988). The aim of the study is to determine the inactivation capacity of propionic, formic and acetic acids on mycelial growth of plant pathogens.

The mentioned organic acids are widely used to prevent the development of plant diseases, to reduce the elongation of the germ tube and to inactivate spore germination. Organic acids show respectable results on the mycelium of soil pathogens and their inactivation (Goepfert and Hicks 1969, Kunte et al. 1998, Sholberg 1998).

Article 5

Effect of acetic acid treatment on the quality of stored carrot (Daucus carota L.) seeds

Hanna Dorna, Agnieszka Rosihska and Dorota Szopihska

Institute of Phytopathology, Seed Science and Technology, Poznań University of Agriculture, Dabrowskicgo 159, 60-594 Poznań, Poland Agronomy 2021, 11 (6), 1176; Post received: April 27, 2021 / Edited: May 19, 2021 / Accepted: June 3, 2021 / Published: June 9, 2021

Source: https://www.mdpi.eom/2073-4395/l 1/6/1176 https://doi.org/10.3390/agronomyl 1061176

4. Discussion

Due to its antifungal and antibacterial properties, acetic acid is commonly used in food production as a preservative and disinfectant [21, 22, 23, 24]. These properties have encouraged

- 12 CZ 38194 U1 scientists who also tried to use it for plant protection, including seed treatment [ 5,6,7,8,9 , 10 , 11 , 12 , 13,25 ]. Various methods of applying acetic acid have been proposed to control seed-borne pathogens, e.g., acetic acid vapor fumigation, mixing seeds with a specific amount of acid, or soaking seeds in acidic solutions.

Our previous experiments, as well as the present results, revealed that soaking in acetic acid solutions effectively controlled Alternaria fungi associated with carrot seeds [13]. In addition, this experiment showed that the positive effect of acetic acid treatment persisted after short-term (5 months) and long-term (12 months) storage.

In general, before and after storage, regardless of temperature, seeds of both samples treated with acetic acid were characterized by a lower infestation of A. alternata and A. radicina than untreated seeds and seeds soaked in distilled water. In addition, acetic acid often controlled these fungi more effectively than fungicide. The treatment was also effective against A. dauci, but the decrease in seed infestation was most pronounced after storage, in the case of sample I, which was characterized by a higher level of initial seed infection by this fungus than sample II.

In general, especially after storage, an increase in acetic acid concentration resulted in a significant increase in the number of mold-free seeds and a decrease in seed germination.

Carrot seeds soaked in 0.5 and 1% acetic acid solution showed higher germination after storage than untreated seeds, while treatment of seeds with 2% acetic acid solution often adversely affected their germination and vigor. Previous experiments have confirmed that too high a concentration of acetic acid can be phytotoxic to plants and seeds.

Pasini et al. [ 26 ], who studied the effectiveness of acetic acid against powdery mildew (Sphaerotheca pannosa var. rosae Woron., syn. Podosphaera pannosa (Wallr.) de Bary) in a greenhouse, found that white vinegar applied at 5 and 10% concentration provided good control disease, but 0.25 and 0.5% acetic acid were phytotoxic.

Sholberg et al. [7] observed that fumigation of wheat seeds with acetic acid vapors at 2 and 4 g/1 kg of seeds controlled common bunting (T. tritici and T. laevis) in the field as effectively as fungicide, but acetic acid applied at a higher dose, i.e. .4 g/1 kg, caused a reduction in the number of sprouts.

Szopiήska [10] reported that soaking in 1, 2.5 and 5% acetic acid solution significantly reduced the infestation of zinnia seeds by Alternaria spp., Cladosporium spp., Fusarium spp. and Gonatobotrys simplex Corda (syn. Melanospora simplex (Corda) D. Hawksw.), but regardless of concentration, treatments reduced the total number of germinating seeds and germination, increased the number of abnormally deformed seedlings, and prolonged germination. Despite these observations, there are reports that properly applied acetic acid treatments can improve seed germination.

El-Saidy and El-Hai [ 11 ] found that soaking sunflower seeds in acetic acid resulted in an increase in seed germination percentage and seed vitality before and after 6 months of storage.

In addition, the treatment reduced the occurrence of several fungal genera, i.e. Alternaria, Aspergillus, Fusarium, Penicillium, Rhizoctonia, Rhizopus, Stemphylium, Trichothecium and Verticillium, before and after storage. In terms of seed storage, seed protection against storage fungi, i.e. Aspergillus spp. and Penicillium spp. is another advantage of acetic acid treatment, as these fungi are considered to be the main cause of seed spoilage.

This possibility was also investigated by Sholberg and Gaunce [ 9 ], who treated canola, maize, rice and wheat seeds artificially inoculated with A. flavus with acetic acid fumes. The authors reported that seed fumigation (crop disinfestation: solution fumigation and steam fumigation - cereal grains, etc.) of wheat with acetic acid at an amount of 0.78 ml/kg of seed resulted in improved seed germination and complete eradication

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A. flavus after 102 days of storage at 20 °C. This fungus was also not detected on canola seeds treated with acetic acid at 0.58 ml/kg seeds after 38 days of storage at the same temperature.

In this experiment, seed-borne fungi were significantly affected by storage temperature. Seeds stored at 4 °C were characterized by higher fungal attack than seeds stored at 20 °C.

Low temperature, optimal for seed storage, usually also promotes pathogen viability [ 27 ]. A decrease in temperature can therefore increase the survival time of seed-borne microorganisms, however, some pathogens, especially in the case of short-term storage, may not respond to this factor [ 28 ].

We observed this phenomenon in sample II after 5 months of storage in relation to the infestation of A. Dauci and A. radicina seeds. However, after 12 months, seeds stored at a lower temperature showed a higher level of infection with these fungi. Alternaria spp. are considered long-lived fungi, and conditions favorable to seed viability also contribute to pathogen survival [ 2,29 ]. Therefore, the possibility of controlling these fungi during storage with an inexpensive and environmentally safe organic compound appears very promising.

Article 6

Acetic acid treatment to maintain the post-harvest quality of Regina and Taloppo table grapes.

T Venditti 1, G D'Hallewin, A Dore, MG Molinu, P Fiori, C Angiolino, M Agabbio Source: https://pubmed.ncbi.nlm.nih.gov/19226763/ PMID: 19226763

The most important postharvest pathogen for table grapes is Botrytis cinerea (gray mold), which causes rapid deterioration of the fruit. Effective disease control during storage is difficult and remains an unsolved problem, as European legislation does not allow any pesticide treatment. GRAS (Generally Recognized as Safe) compounds, used without restriction as preservatives in Europe and North America, are possible candidates to bridge this gap. The aim of this work is to study the effectiveness of Acetic Acid (AAC - acidum aceticum), used as a post-harvest treatment to control Botrytis cinerea on Regina and Taloppo table grapes, through laboratory and storage tests. The activity of this compound was first evaluated by laboratory tests in which individual berries were inoculated with B. cinerea at different concentrations (0, 5, 10, 20, 50, 75 and 100 microl/1) of AAC vapor for 15 minutes.

After treatment, the fruit was incubated at 20°C for one week. The in vivo experiment was conducted using the most promising concentrations of AAC (50, 75 and 100 microl/1), followed by eight weeks of storage at 5 degrees C and 95% relative humidity (RH) and four days at 20 degrees C and 85% RH (simulated shelf life conditions).

At the end of the in vivo experiment, weight loss and visual assessment were evaluated. Almost all treatments reduced gray mold after eight weeks of storage. The best results were obtained with the use of 50 ppm AAC, achieving a reduction in spoilage compared to untreated Taloppo and Regina grapes of 61.0% and 41.4%, respectively. After the simulated shelf life, the differences between treated and untreated (control) became insignificant for Taloppo grapes, while the lowest percent decomposition was achieved with 50 microl/1 AAC for Regina grapes (52% reduction compared to control). Regarding fruit weight loss, none of the treatments significantly affected this parameter, which ranged between 8.2% and 11.5% after eight weeks of storage and 13.5% and 18.2% after shelf life. At the end of storage, the highest visual score was assigned to the fruit treated with 50 microl/1 AAC, demonstrating a clear better storability During this period, slight damage was observed on the berries after the application of AAC at 75 and 100 microl/1. The reported results obtained from these experiments indicated that acetic acid could be a promising compound for use as an alternative to SO2

- 14 CZ 38194 U1 (Sulfur oxide) in maintaining grape quality and controlling rot during storage.

Article 7

Fumigation of fruit with acetic acid to prevent decay after harvest

P. L. Sholberg and A.P. Gaunce

Agriculture and Agri-Food Canada, Research Centre, Summerland, British Columbia, V0H 1Z0, Canada

Additional index words. Botrytis cinerea, Penicillium expansum, apple, steam, volatiles Source: https://j ournals.ashs. org/hortsci/view/journals/hortsci/30/6/article-p1271.xml

Abstract.

Acetic acid (AA, Acetic Acid ) as a vapor in low concentrations was effective in preventing fruit from decay by postharvest fungi. Fumigation with 2.7 or 5.4 mg AA/liter in air at 2 and 20 °C reduced germination of Botrytis cinerea Pers. and Penicillium expansum after being dried on 0.5 cm square pieces of dialysis tubing. Decay, inoculated Delicious', 'Red Delicious' and 'Spartan' apples (Malus domestica Borkh.) with 20 μ! drops of B. cinerea conidia (1.0 ^x 105 conidia/ml) or P. expansum (1.0 ^x 106 conidia/ml) were prevented by fumigation with 2.0 and 2.7 mg AA/liter, respectively.

Tomatoes (Lycopersicon esculentum Mill.), grapes (Vitis vinifera L.) and kiwi [Actinidia deliciosa (A. Chev.) C.F. Liang et R. Ferguson var. deliciosa] inoculated with B. cinerea or oranges (Citrus sinensis L.) inoculated with P. italicum Wehmer did not decay when fumigated with 2.0 mg AA/liter at 5°C.

AA fumigation at low temperatures (1 and 5C) with 2.0 or 4.0 mg AA/liter prevents rotting of 'Spartan' and 'Red Delicious' apples and 'Anjou' pears (Pyrus communis L.) inoculated with B. cinerea and P .Expansion. 'Spartan' apples dipped in a suspension of P. expansum conidia (1.4 ^x 105 conidia/ml) and fumigated with 2.7 mg AA/litre at 5°C had an average of 0.7 lesions per fruit compared to up to 6.1 for untreated fruit.

Increasing relative humidity from 17% to 98% increased AA fumigation efficiency at 5 and 20 °C. At the concentrations used in our tests, AA had no apparent phytotoxic effects on fruit.

The potential for commercial use is emerging when fumigation shows promise with AA to control postharvest decay of fruits and vegetables.

Article 8

Fumigation of stone fruits with acetic acid to control postharvest decay

Crop Protection Volume 15 Issue 8 December 1996 Pages 681-686

Peter L. Sholberg, Alan P. Gaunce

Source: https://www.sciencedirect.com/science/article/abs/pii/S0261219496000397

Summary:

Acetic acid was an effective post-harvest fumigant to kill mold spores on peaches, nectarines, apricots and cherries.

The decay of Monilinia fructicola and Rhizopus stolonfer on Harbrite peaches was prevented only by 1.4 and 2.7 mg/l of acetic acid.

Harbrite peaches fumigated with 2.7 mg/L acetic acid were slightly injured, phytotoxicity indicated by light brown streaks.

Higher concentrations of acetic acid increased injury; the streaks darkened and became much more pronounced.

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Glohaven peaches treated in the orchard with captan at 5% bloom, full bloom, fruit ripening and 2 days before harvest then fumigated with 2.7 mg/l acetic acid postharvest had significantly less postharvest brown rot (12.5%) than fruit treated with captan alone (25.0%).

Decay of Lambert cherries, primarily due to Alternaria spp., was reduced from 38.9 to 10.0% by fumigation with 2.7 mg/L acetic acid.

Unfortunately, during storage at 1 °C, small pits formed on the surface of the fruits. Brown rot (M. fructicola) of Tilton apricots was reduced from 100 to 25% by fumigation with 2.0 mg/L acetic acid without evidence of severe phytotoxicity.

Article 9 (from PDF)

Evaluation of L-ascorbic acid (VITAMIN C) IN VITRO, against Monilinia fructigena (Fruit nematode), Alternaria solani (Alternaria spot of tomato) and VENTURIA INAEQUALIS (Apple scab - Apple scab) - Donyo H. Ganchev

Agricultural University - Plovdiv AGRICULTURAL SCIENCES Volume 14, Issue 35, 2022 Article in Agricultural Sciences^February 2023 DOI: 10.22620/agrisci.2022.35.006 Source: http://agraminauki.au-plovdiv.bg/2022/issue-35/6 -35/

Summary:

In vitro antifungal tests with one of the most popular medicinal substances in the world: L - ascorbic acid, widely known as vitamin C, were carried out with the three most widespread plant pathogens in Bulgaria: Monilia fructigena, Alternaria solani and Venturia inaequalis in order to evaluate the direct antifungal action of the acid used in agriculture as an ISR promoter (phytovaccine). Vitamin C therefore has strong antimicrobial properties that reduce the pathogenicity of bacteria, viruses, parasites and fungi. The tests performed showed a strong antifungal activity of L-ascorbic acid against the mycelium and conidiospores of Alternaria solani and Venturia inaequalis and a very weak antifungal effect against Monilia frucigena, which means that vitamin C can be used as a direct fungicide and phytovaccine in the protection against pests. at the same time. The expression of pesticidal action of ISR promoters is not uncommon in contrast to animal and human vaccines.

In recent years, the area of increased interest in l-ascorbic acid is also pest control. Vitamin C, along with salicylic acid, has been found to be a key component of the so-called oxygen burst, which is an essential part of the system-activated resistance (SAR) and induced-systemic resistance (ISR) in plants (Davey et al., 2000; Khan et al. , 2011; Boubakri, 2017). Other research shows strong stimulatory effects of the substance on the growth and development of plants and their resistance to adverse abiotic conditions such as drought and especially cold (Guo et al., 2005; Gallie, 2013; Kaur & Nayyar, 2014).

There is a growing number of agrochemicals - products that contain vitamin C as an active ingredient, e.g.:

- SNS-DCtm fungicide - contains active substances: L-ascorbic acid, silicon dioxide, potassium sorbate and soap bark

- Sunonic® broad-spectrum fungicide and bactericide - contain active substances: L-ascorbic acid, ethyl lactate, glycerin, sodium chloride.

- Biolife fungicide, bactericide and virucide - contain active substances: L-ascorbic acid, citric acid, lactic acid.

Conclusion:

The current study shows that the substance may have a direct antifungal effect against some plant pathogens. However, there is a great difference of this antifungal with respect to different pathogens. The acid showed very weak antifungal activity against Monilia fructigena, yet

- 16 CZ 38194 U1 cause more phytotoxicity on cherries, which are very often affected by the pathogen Monilia fructigena. According to Alternaria solani and Venturia inaequlis, vitamin C can be used as a safe, cheap and natural alternative against commercial fungicides. Expression and pesticidal action of ISR promoters like vitamin C is not uncommon, unlike animal and human vaccines. The solubility of vitamin C in water is good and thus vit. C very suitable for formulation as a plant protection product due to the ability to form true solutions without sedimentation or separation.

Article 10 (from PDF)

National Center for Biotechnology Information

Salicylic acid (SA - salicylic acid) fights Fusarium wilt by inhibiting the target of rapamycin signaling pathway in Fusarium oxysporum

Linxuan Li, a, b, c, 1 Tingting Zhu, a, b, c,1 Yun Song, d, 1 Li Feng, a, b, c Philip James Kear e Rooallah Saberi Riseh, f Mahmoud Sitohy, g Raju Datla, ha Maozhi Ren a, b, c,*

J Adv Res. July 2022; 39: 1-13. PMCID: PMC9263656

Published online November 2, 2021 doi: 10.1016/j.jare.2021.10.014 PMID: 35777900

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9263656/

Summary - Biofungicides with low toxicity and high efficacy are a global priority for sustainable agricultural development. The phytohormone salicylic acid (SA) is an ancient drug against various diseases in humans and activates the plant immune system, but little is known about its function as a biofungicide.

Objectives - Here, Fusarium oxysporum, the causative agent of Fusarium wilt and immunosuppressive "patients", was used as a model system to investigate whether SA can enter pathogen cells and suppress key pathogen targets.

Methods - Oxford Nanopore MinION sequencing and high-throughput chromosome conformation capture (Hi-C) sequencing were used to analyze the Fusarium oxysporum genome. In addition, RNA-seq, qRT-PCR and western blotting were performed to detect gene and protein expression levels.

Result - From potato dry rot, we isolated and sequenced the genome of F. oxysporum and Fusarium oxysporum, which included 12 chromosomes and a genome length of 52.3 Mb. Pharmacological tests showed that exogenous application of SA can effectively stop the growth of hyphae (fungal filaments), spore production and pathogenicity of F. oxysporum, while endogenous salicylate hydroxylases significantly detoxify SA. Synergistic inhibition of F. Oxysporum growth was observed when SA was combined with rapamycin. Kinase assays showed that SA inhibits FoTOR complex 1 (FoTORCl) by activating FoSNF1 in vivo. Transgenic potato plants with FoTOR1 and FoSAH1 gene interference inhibited invasive hyphal growth and significantly prevented the occurrence of Fusarium wilt. In addition, previous studies have shown that SA enhances antifungal activity by inhibiting hyphal growth and spore germination in Fusarium oxysporum, Magnaporthe grisea, and Penicillium expansum.

Conclusion

This study revealed the underlying mechanisms of SA against F. Oxysporum provided insight into SA in controlling various fungal diseases by targeting the SNF1-TORC1 pathway of pathogens.

Article 11

Abiotic stress tolerance in plants mediated by citric acid

According to Md. Tahjib-Ul-Arif 1,2,*, Revenge. Ishrat Zahan 3,f, Md. Masudul Karim 4,f, Shahin Imran 5, Charles T. Hunter 6, Md. Saiful Islam 7, Mrs. Ashik Mia 4, Md. Abdul Hannan 2, Mohammad Saidur Rhaman 8, Md. Afzal Hossain 2, Marian Brestic 9,10, Milan Skalický 10a, Yoshiyuki Murata

1. Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan

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2. Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

3. Plant Breeding Division, Bangladesh Rice Research Institute, Gazipur 1701, Bangladesh

4. Department of Plant Botany, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh 5. Department of Agronomy, Khulna Agricultural University, Khulna 9100, Bangladesh

6. Chemical Research Unit, United States Department of Agriculture - Agricultural Research Service, Gainesville, FL 32608, USA

7. Department of Fisheries, Bangamata Sheikh Fojilatunnesa Mujib Science and Technology University, Melandah, Jamalpur 2012, Bangladesh

8. Department of Seed Science and Technology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

9. Department of Plant Physiology, Slovak University of Economics, 94976 Nitra, Slovakia 10. Department of Botany and Plant Physiology, Faculty of Agrobiology, Food and Natural Resources, Czech University of Economics, Prague, 16500 Prague, Czech Republic

International J. Mol. Sci. 2021, 22 (13), 7235; https://doi.org/10.3390/ijms22137235

Article received: 5 June 2021 / Edited: 26 June 2021 / Accepted: 27 June 2021 Published: 5 July 2021 (This article is under Molecular Plant Sciences)

Source: https://www.mdpi.com/1422-0067/22/13/7235#

Summary

Several recent studies have shown that citric acid (CA) can confer tolerance to abiotic stress in plants. Exogenous application of CA leads to improved growth and yield in crops under various abiotic stress conditions. Improved physiological outcomes are associated with higher photosynthetic rates, reduced reactive oxygen species and better osmoregulation. CA application also induces antioxidant defense systems, promotes increased chlorophyll content, and affects secondary metabolism to limit plant growth limitation under stress. In particular, CA has a great effect on alleviating heavy metal stress by promoting the precipitation, chelation and sequestration of metal ions. This review summarizes the mechanisms that mediate CA-regulated changes in plants, particularly the involvement of CAs in the control of physiological and molecular processes in plants under abiotic stress conditions. We also review genetic engineering strategies for CA-mediated abiotic stress tolerance. Finally, we propose a model that explains how CA's position in complex metabolic networks involving the biosynthesis of phytohormones, amino acids, signaling molecules and other secondary metabolites could explain some of its abiotic stress-relieving properties. This review summarizes our current understanding of CA-mediated abiotic stress tolerance and highlights areas where further research is needed.

3. Exogenous CA to alleviate abiotic stress

3.1. Salinity stress

Exogenous (external) application of citric acid/CA can increase plant tolerance to salinity and ultimately increase growth and yield (Table 2). Carica papaya (papaya) seeds activated with CA solution showed improved germination under salt stress conditions [ 42 ]. Foliar spray with CA reduced the sensitivity of G. Barbadense to salt stress, improved growth and yield and resulted in higher total soluble sugars (TSS), total soluble protein (TSP), total phenolic compounds (TPC), free amino acids (FAA) and proline content [ 18 ]. In addition, El-Hawary and Nashed [ 43 ] reported that foliar application of CA combined with ascorbic or salicylic acid increased the growth and productivity of maize under salt stress conditions. Application of CA in combination with ascorbic acid and thiamine improved salinity tolerance by up-regulating non-enzymatic antioxidants (TPC and proline accumulation) and decreasing enzymatic antioxidants [CAT, POX and phenylalanine ammonia lyase (PAL)] in H. sabdarfa and Melissa oficinalis (lemongrass), a response related to maintaining the cellular redox state [ 44,45 ]. Several studies have shown that

- 18 CZ 38194 U1 application of CA can increase the activity of antioxidants, including superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), glutathione peroxidase (GPX), polyphenol oxidase (PPO) and ascorbate peroxidase (APX). cotton, maize, Beta vulgaris (sugar beet), Hibiscus sabdarfa and Leymus chinensis (Chinese ryegrass) [ 18,43,45,46,47 ]. Essential oil components (monoterpene hydrocarbons and oxygenated sesquiterpenes) of lemon balm under salt stress conditions were increased by CA treatment [ 44 ]. Application of CA in sugar beet alone or in combination with banana and/or tomato peel extracts improved root yield in saline soil [ 47 ].

The essence of the technical solution

The goal is to shed light on the understanding of the factors influencing the formulation of the Bio-fungicide presented by us (based on natural substances), for use in agricultural and natural ecosystems (including so-called crop fumigation). It is a pre- and post-infection fungicide - pre-harvest for plants and post-harvest for the so-called fUmigation. The use of these technologies, especially in agricultural production and the innovative power of these technologies, is able to develop its potential and limit the inappropriate action of synthetic fungicides in agriculture. These new natural-based technologies not only have great potential to increase food production globally, but also to improve its quality and reduce the effects of excessive intensification of agricultural production.

A bio-fungicide that we formulate on the basis of effective inhibition of the growth and development of pathogens in plants and in the soil. The product helps increase crop productivity by reducing competition between plants for nutrients, water and light. Their ecological nature distinguishes this Bio-fungicide from synthetic fungicides by supporting ecological agricultural practices, without synthetic chemicals with impacts on humans, soil and nature as a whole.

Despite their numerous advantages, many common Bio-fungicidal compounds against synthetic fungicides are inherently unstable and prone to degradation in environments with higher temperature, light, humidity and microbial activity, posing a challenge for new effective plant pathogen management.

To solve this problem, in recent years, more and more emphasis has been placed on strategies for using substances with lower degradability, which we achieve by using various organic acids.

Biofungicides consist of microorganisms or natural compounds and are used for foliar diseases of plants, for soil treatment and also for post-harvest applications on crops (so-called fumigation).

Therefore, antimicrobials and antiseptics, including surfactants, can help improve fungicide efficacy by increasing their effectiveness and bioavailability, reducing the amount of product needed for treatment, and increasing their ability to target plant infestations while preserving the crop.

However, it is important to choose the right materials, depending on specific needs and consideration of several factors inherent to surfactants and other substances, such as production costs, etc.

The aim of this preparation is to provide a safe non-toxic Bio-fungicidal composition that does not harm the environment.

The presented technical solution is focused on a fungicidal preparation containing in particular - Acetic acid 10% (organic acid), with other active substances and emulsifiers, for the control of plant diseases. The preparation is intended for its activity to kill pathogens in plants and in the soil, it is also intended for post-harvest fumigation, while it is a contact fungicide.

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In our case, we use this formulation:

The active substance - acetic acid 10%, other natural substances - citric acid to create a synergistic effect together with salicylic acid and L-ascorbic acid, which have a high ability to inhibit many fungi (overview of inhibited pathogens, microorganisms - see below).

The key hormone released during plant defense is the Salicylic Acid (salicylate) reaction. Furthermore, the formulation contains an adjuvant/surfactant - Glycerin, a natural surfactant/wetting agent, etc.

Acetic acid (or White Vinegar 10%) - is present in a low concentration that is allowed by the EU. For a more effective combination of the fungicidal preparation, we chose the surfactant Glycerin - an organic substance, while it is a natural, surface-active substance. This natural non-toxic compound can be used as a safe alternative for pathogen control in organic farming systems.

Plants are constantly in danger of damage to their organs by animals but also by many types of microorganisms. Therefore, in addition to a large number of morphological and morphogenetic adaptations, they also have diverse biochemical adaptations.

Therefore, the goal of this combined preparation is also the use of salicylate in plant protection with our presented formulation of this preparation, for the defense reaction of plants. In this formulation, we place particular emphasis on the already mentioned properties - acetic and citric acids, as well as salicylic acid and L-ascorbic acid. Emphasis is also placed on the synergistic activity of another substance with surfactant functions, i.e. surfactants that reduce the surface tension on the leaf, namely Glycerin, which also reduces the amount of fungicide used during spraying, thanks to its complex adjuvant function.

This means that Glycerin is a so-called synergist multiplying the effect of acetic acid and at the same time by reducing the amount of the product used when spraying due to the wetting agent (Glycerin) used, among other things by limiting the evaporation of active substances from the leaf, when applying the product.

The presented technical solution provides a kind of effective natural fungicidal preparation for killing pathogens, but also more environmentally friendly, which can be used to improve the control of the effect of microorganisms and adapts to current ecological requirements.

Acetic acid in a concentration of 10% vol. (so-called white vinegar - food grade)

It is essentially a 10% fermented food grade vinegar, which is produced by a natural fermentation process of alcohol using vinegar bacteria, especially of the genus Acetobacter. It is a genus of aerobic bacteria from the Alphaproteobacteria group, which are obtained by oxidizing alcohol to acetic acid. Compared to ordinary 5 to 8% vinegar, it is more concentrated and is not colored with caramel E150c. Therefore, it has a white transparent color and is sometimes called white vinegar or colorless vinegar (density - 1.0125 g/cm ³ ).

For the fungicide product, it is necessary to choose vinegar with a higher concentration, i.e. vinegar (food grade with a maximum 10% acetic acid content), which is approved by ÚKZÚZ (Central Control and Testing Institute for Agriculture Brno) as a plant protection agent, for use as a fungicide . This vinegar must be used in accordance with the special conditions of the EU.

The mentioned organic acids are widely used to prevent the development of plant diseases, e.g. to reduce germ tube elongation and inactivate spore germination. Organic acids also show respectable results on the mycelium of soil pathogens and their inactivation. The product can be applied to plants/crops as well as to soil to reduce pathogen populations.

This substance - a natural non-toxic preparation, can be used as a safe alternative for control

- 20 CZ 38194 U1 fungicides in organic farming systems, given that Acetic acid 10% and other substances are of particular interest to companies that are under pressure to use more environmentally friendly chemicals - especially those that are certified according to ISO 14001:2016 ( ISO 14001 is a globally recognized standard for environmental management systems - EMS, which was first published in 1996).

Ethanoic acid (vinegar) in a concentration of 10% is approved as a weed killer that does not translocate to plant roots, whereas e.g. the more concentrated ethanoic (acetic) acid 20% must not be offered as a weed control product due to EU-ECHA regulations.

- acetic (ethanoic) acid is valued in agriculture for its natural fungicidal properties

- used as an alternative to synthetic chemicals that can have negative effects on the environment

- can be applied to plants and soil to reduce pathogen populations

- acts against a wide range of molds and fungi that attack plants

- acetic acid is effective against fungi that cause rotting of fruits and vegetables

- helps control mold in the soil, which improves crop health and yield

- the use of acetic acid can lead to an increase in soil biodiversity

- acts as a natural inhibitor of mold growth without negative side effects

- application of acetic acid can reduce the need for expensive synthetic pesticides/fungicides

- acetic acid is safe for the environment because it quickly decomposes into harmless substances

- can be used in organic farming, which requires the use of natural resources

- farmers can easily apply acetic acid using sprayers

- its effectiveness can be increased by combining it with other natural substances

- acetic acid can also be used to disinfect agricultural tools and equipment

- the use of acetic acid can improve the durability and quality of harvested crops

- application to seeds before planting can help protect young plants from fungi

- acetic acid is also useful for preventing mold during crop storage

- when used correctly, acetic acid does not cause any damage to plants

- helps to maintain a clean and healthy environment in greenhouses and foil houses

- it can also be used to treat compost to prevent mold growth

- acetic acid can contribute to improving the overall quality of the soil

- its low toxicity guarantees that it will not endanger the health of farmers or consumers

- with the correct dosage, acetic acid does not leave any harmful residues

- the use of acetic acid can support the sustainability of agricultural practices

- acetic acid can be part of biological plant protection programs

- its effectiveness is proven in various climatic conditions

Citric acid (food grade)

Citric acid is an organic compound with the chemical formula C6H8O7. It is a colorless organic acid. Citric acid can be obtained as an anhydrous form or as a monohydrate. The anhydrous form crystallizes from hot water, while the monohydrate is formed when citric acid crystallizes from cold water.

In 1917, the American food chemist James Currie discovered that certain strains of Aspergillus niger could be efficient producers of citric acid. In biochemistry, it is an intermediate in the citric acid cycle that occurs in the metabolism of all aerobic organisms.

Since citric acid inhibits some types of bacteria and viruses, you can find it e.g. in products that kill fungi or algae, in disinfectants, etc. It is also used as a disinfectant, virucidal and germicide.

Citric acid is the active ingredient in various pesticides for residential and commercial use such as

- 21 CZ 38194 U1 disinfectants and fungicides. These products contain citric acid, even in combination with other active substances.

Salicylic acid

Salicylic acid has long been a key starting material for the production of acetylsalicylic acid (ASA or Aspirin). Salicylic acid modulates the enzymatic activity of COX-1 and reduces the production of anti-inflammatory prostaglandins, while it is also an extract from willow bark, called Salicin (after the Latin name for white willow).

Salicylic acid occurs in plants as - free salicylic acid and its carboxylated esters and phenolic glycosides. Salicylic acid is the so-called a phytohormone and at the same time an antifungal agent found in plants with certain functions in plant growth and development, i.e. - photosynthesis, transpiration and uptake of ion transport. Salicylic acid participates in endogenous (internal, in the given organism) signaling, whereby it mediates the defense of plants against various pathogens.

Salicylic acid is also very important in resistance to pathogens (i.e. systemic acquired resistance of plants) by inducing the production of proteins related to pathogenesis - the development of various plant diseases and other defensive metabolites.

L-ascorbic acid

L-ascorbic acid, or vitamin C, is a water-soluble organic compound, in a solid crystalline state, with a white appearance. It is a basic substance, very important for the human organism and at the same time it is also important for plants.

L-ascorbic acid functions as the main redox buffer (conjugated acid pair that is able to maintain a stable pH within a certain range) and as a cofactor for enzymes involved in the regulation of photosynthesis, hormone biosynthesis and the regeneration of other antioxidants. This acid is not only an antioxidant, but is necessary for photosynthesis and at the same time has a role in the regulation of cell division and also flowering, while, as already said, it acts as a cofactor in many enzymatic reactions, which is essential for the overall outcome of the given effects.

The role of ascorbic acid in defense against pathogens has received only limited attention. In studies to date, the content of L-ascorbic acid led to a reduction in the growth of the bacterial pathogen when Arabidopsis vtc 1 (mutant) or vtc2 was attacked by pathogens, as well as in Pseudomonas syringa pv maculicola and inhibition of hyphal growth of the fungal pathogen Peronospora parasitica.

This means that an increase in the content of L-ascorbic acid can affect the defense reactions of plants. The function of L-ascorbic acid is integrated into plant growth and development and its importance cannot be underestimated (e.g. it can inhibit the growth of fungal cultures according to research).

In the future, research strategies may include highly sophisticated approaches to alter the content of L-ascorbic acid in specific cell types or tissues, in order to achieve the desired goal in bio-fungicide plant treatment, which is also our goal.

Glycerin (vegetable glycerol 99.5% by volume)

Glycerin (or glycerol) is a natural substance - adjuvant, surfactant, surfactant, anionic surfactant and also an activator. Glycerin (organosilicone surfactant - density 1.25 g/cm ³ ) sticks better to the leaf thanks to its properties, it is also a vehicle - a carrier, i.e. a substance/matter added to an active substance that supports changes in their physicochemical properties (synergistic substance ). At the same time, it acts as an emulsifying moisturizer - emulsifier and agent. Glycerin is also a non-ionic surface-active agent that can quickly reduce the surface tension of the applied substance on the leaf blade, and its strong wettability promotes the spreading ability of the substance Acids

- 22 CZ 38194 U1 acetic 10% etc., on the head of the leaf blades.

This substance helps the spray solution to wet the surface of the plants, thereby helping to strengthen the active substance (acetic acid 10%). That is, it is a synergist that simultaneously functions as a universal adjuvant - carrier, moisturizer (it also serves as an antifreeze agent).

It is a colorless, odorless, viscous liquid. This organic compound can quickly reduce the surface tension of the preparation on the leaves, and at the same time, its wettability, serves as a surfactant for the use of the overall fungicidal preparation, with the fact that after homogenization with the other ingredients of the preparation, it is a slightly emulsifiable liquid (glycerin - emulsifier).

Glycerin is also an activator - they increase the wettability and thus the adhesion (adhesion) of the application liquid and its permeation (penetration) properties, while it can stimulate the conductive transport of acetic and citric acid in the plant body and, based on this, improve (strengthen) the effect of the product.

Glycerin (surfactant) in larger quantities will use its own ability to maintain moisture on the leaf, which means that both acetic acid and citric acid are absorbed, so that the leaf absorbs the relevant substances for a longer period and thus reduces the dosage of the product as a whole.

This improves the overall effect of the biofungicide product in question and also extends the time effectiveness of the product thanks to Glycerin.

The spray volume can be reduced by up to 70% when using Glycerin, to reduce water consumption and the cost of surfactant application. In particular, the total volume of the fungicide preparation is reduced during the spraying itself. This means that Glycerin (in this case a glycerin synergist) serves not only as a powerful wetting adjuvant, which can significantly improve the penetration of fungicidal liquid under the surface of weed leaves, as well as improve the target absorption and transport of fungicidal active ingredients, in favor of reducing the use of the amount of herbicide as a whole when spraying.

According to this composition containing glycerin, the application amount of the active substance - Acetic acid 10% is sufficient (after diluting the concentrated preparation), while the preventive effect is improved, while it is a so-called synergistic effect for the effective effectiveness of the presented biofungicidal composition. It is well known that there is an inverse correlation between dynamic surface tension and leaf retention with Glycerin in agricultural spray solutions.

The preparation should meet the following criteria for a fungicidal preparation as part of the technical solution:

- fungicide use on plants and soil, including post-harvest fumigation on stored crops

- applying the product to the soil as well, in order to reduce the population of pathogens

- using only natural substances - applies to both acetic and citric acids, salicylic acid, L-ascorbic acid and glycerin

- synergistic effect (natural substances of Glycerin), strengthening the effect of Acetic Acid by facilitating and increasing emulsification (on the leaf there is a force action on the phase interface and thus the formation of small oil droplets in water, when the surface of the oil phase increases) and dispersion (dispersion ) when sticking to the leaf, including subsequent adhesion and thus wetting the surface of the leaf blade. This substance (Glycerin is also a surfactant) will allow the fungicide to be deposited closer to the "skin" of the plants, thereby increasing contact with the leaf and keeping the product in a soluble form, while having the potential to achieve a higher absorption of Acetic Acid.

- higher efficiency due to other substances - Citric acid, Salicylic acid, L-ascorbic acid and Glycerin, due to synergistic effects

- Glycerin is also an adjuvant (substance for exceptional coverage of crop and plant surfaces), which reduces the volume of spraying by up to 70%

- faster degradation of the product in the given environment after application of the product, which is composed only of natural substances, against synthetic fungicides

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Adjuvant - surfactants (surfactants):

They are designed to improve the biological and other factors of this fungicidal composition. The effect of the surfactant Glycerin on the reception of fungicides is significant, especially when applying more expensive preparations, which is very economical as they allow a significant reduction in the dose per unit area without reducing the biological activity of the preparation.

Spray retention (retention of the substance on the leaf) using surfactants is an important parameter of the effectiveness of the fungicide, because they determine the maximum amount of the product that can penetrate the surface of the plant leaf. The higher the amount of retained fungicide, the smaller the contact angle the droplets have on the leaf surface and can expand, thereby achieving a larger leaf contact surface. Plant species show different wettability on their leaves, due to different leaf architecture such as leaf roughness, surface "wax" on the leaf, etc.

Based on this, the surfaces of plant species can be classified from easily wettable to difficult to wettable, which describes why some plants exhibit very non-wetting properties and lead to low retention of sprayed liquids.

This means that for a better use of the fungicide product, the addition of various types of adjuvants, surface-active substances, could be beneficial not only for the effectiveness of the fungicide but also for the environment, thanks to the lower applied doses of the presented product.

However, if the surface of the solution increases (the formation of spray droplets), the need for additional surfactant molecules at the surface interface arises, and if there is not a sufficient concentration of surfactant in the solution, the reduction of surface tension may not be maximally possible. Often, the amount of wetting agent added to the application solution exceeds the value of KMK (critical micellar concentration) - according to measurements of conductivity and surface tension of solutions, exceeding KMK.

This point is the critical micellar concentration (CMC), further increasing the wetting agent concentration in the solution no longer reduces the surface tension, but the wetting agent molecules aggregate and form various aggregates and micelles (surfactant molecules are so-called hydrophilic and oleophilic, which means that they attract water, grease and dirt).

In our case, the surfactant (Glycerin) does not really change up to the weight percentage of glycerin, equal to 20% w/w, according to research.

The most important group of adjuvants are wetting agents - they are substances that increase the biological effectiveness of fungicides by increasing and accelerating the penetration (also called penetrants) of substances into leaf tissues. Wetting agent molecules are amphipathic (ie, they consist of two parts), each of which is attracted to a different phase. The front part of the molecule is usually hydrophilic and the end part is lipophilic and thus forms a kind of bridge between the two phases, thereby reducing the surface tension of the spray droplets.

Bio-fungicides are applied to plants and have little or no residual activity in the soil, meaning we can apply them multiple times as needed.

According to the United States Department of Agriculture (USDA), glycerin has excellent antibacterial, antifungal and antiviral properties. It is also a solvent and preservative in skin care products that contain herbal extracts.

When using this herbicide, according to the technical solution, we aim to achieve the following synergistic effects between the individual chemical substances on the leaf blade, with the following:

1. We use Glycerin to expand the effective contact area of the drops, reduce the air gap

- 24 CZ 38194 U1 between the solution and the surface of the leaf blade, while reducing the anti-diffusibility of the cuticle of the blade (cuticle in plants - it is a protective hydrophobic waxy covering of the leaf).

2. Glycerin is fundamentally involved in wetting, it prevents the drops of the product from drying quickly, thereby extending the wetting time, i.e. penetration of substances into the leaf and thereby prolonging the reaction of the product.

3. We activate chemicals entering the leaf blade of the plant when the chemicals infiltrate the cuticle, as a co-solvent or stabilizing agent that affects solubility, i.e. we cause infiltration of the active substance and also other substances.

Furthermore, with this procedure (the composition of the fungicide), we try to better adapt to the relevant national requirements for environmental protection.

For this reason, the substances contained in the formulation can reduce the amount of fungicide used during the application itself (application to plants by spraying) and increase the effect of this preparation, based on the synergists - Citric acid, Salicylic acid, L-ascorbic acid and Glycerin.

Examples of implementing a technical solution

It is a composition of a fungicidal preparation on a natural basis (pre- and post-infectious fungicide - pre-harvest for plants and post-harvest for the so-called fumigation of crops) - Acetic acid (the main active ingredient), which acts with its antifungal activity on plants, with the use of other synergistic substances , adjuvants, etc. The presented examples of implementation of the technical solution provide a kind of effective fungicidal preparation for plants, including post-harvest application to various stored crops (fumigation - treatment of crops by wet means, possibly steam) and also for soil treatment.

The fungicide preparation is diluted according to the given ratio and is prepared in commercial packaging for dilution and subsequent use for the purpose of spraying plants, i.e. protection against pathogens (see below for further details).

This preparation is presented in three examples of the technical solution, including the relevant table.

Example 1

Preparation with subsequent composition of a natural bio-fungicide preparation, to treat plants, etc., against various types of pathogens.

All listed substances/chemicals are in liquid form, or they are soluble in cold water. The substances below were mixed at temperatures between 19 and 23°C.

The raw materials (chemicals) listed in the table of examples were dissolved at room temperature in 10% acetic acid (food grade) to form a fungicidal solution. Tested samples are considered 100% and were diluted in Acetic Acid 10%, to the required concentrations.

The fungicidal composition is prepared using prepared acetic acid (so-called white vinegar in a concentration of 10%), in this case it is a ready-made - purchased solution in which other substances are diluted and is suitable according to valid standards and regulations for the purpose of a natural fungicidal agent.

The following was added to the acetic acid solution (so-called white vinegar with a concentration of 10%): Citric acid - 2.5 g/1000 g, Salicylic acid (Salicyl acid) - 2 g/1000 g, L-ascorbic acid (L-ascorbic acid ) - 3 g/1000, Glycerin (vegetable glycerol) - 52 g/1000 g. The result of this process is a clear homogeneous solution.

- 25 CZ 38194 U1

The balance of up to 100% of the weight of this fungicidal preparation consists of - Acetic acid (so-called white vinegar with 10% concentration).

Example 2

Preparation with subsequent composition of a natural bio-fungicide preparation, to treat plants, etc., against various types of pathogens.

All listed substances/chemicals are in liquid form, or they are soluble in cold water. The substances below were mixed at temperatures between 19 and 23°C.

The raw materials (chemicals) listed in the table of examples were dissolved at room temperature in 10% acetic acid (food grade) to form a fungicidal solution. Tested samples are considered 100% and were diluted in Acetic Acid 10%, to the required concentrations.

The fungicidal composition is prepared using prepared acetic acid (so-called white vinegar in a concentration of 10%), in this case it is a ready-made - purchased solution in which other substances are diluted and is suitable according to valid standards and regulations for the purpose of a natural fungicidal agent.

The following was added to the acetic acid solution (so-called white vinegar with a concentration of 10%): Citric acid - 6 g/1000 g, Salicylic acid - 4 g/1000 g, L-ascorbic acid (Lascorbic aid) - 4 g /1000, Glycerin (vegetable glycerol) - 89 g/1000 g. The result of this process is a clear homogeneous solution.

The balance of up to 100% of the weight of this fungicidal preparation consists of - Acetic acid (so-called white vinegar with 10% concentration).

Example 3

Preparation with subsequent composition of a natural bio-fungicide preparation, to treat plants, etc., against various types of pathogens.

All listed substances/chemicals are in liquid form, or they are soluble in cold water. The substances below were mixed at temperatures between 19 and 23°C.

The raw materials (chemicals) listed in the table of examples were dissolved at room temperature in 10% acetic acid (food grade) to form a fungicidal solution. Tested samples are considered 100% and were diluted in Acetic Acid 10%, to the required concentrations.

The fungicidal composition is prepared using prepared acetic acid (so-called white vinegar in a concentration of 10%), in this case it is a ready-made - purchased solution in which other substances are diluted and is suitable according to valid standards and regulations for the purpose of a natural fungicidal agent.

The following was added to the solution of acetic acid (so-called white vinegar with a concentration of 10%): Citric acid - 5 g/1000 g, Salicylic acid (Salicyl acid) - 5 g/1000 g, Lascorbic acid (L-ascorbic aid) - 6 g /1000, Glycerin (vegetable glycerol) - 93 g/1000 g. The result of this process is a clear homogeneous solution.

The balance of up to 100% of the weight of this fungicidal preparation consists of - Acetic acid (so-called white vinegar with 10% concentration).

- 26 CZ 38194 U1

Based on the given table of examples, the optimal composition from column 3 was selected.

Raw material/substance (g/1000 g) concentrate for dilution 1 2 3 Citric acid 2.5 6 5 Salicylic acid (Salicyl acid) 2 4 5 L-ascorbic acid 3 4 6 Glycerin (vegetable glycerol) 52 89 93 Acetic acid 10% (Ethanoic acid / White vinegar) Up to 1000g Up to 1000g Up to 1000g

The bio-fungicide that we present belongs to the natural complex fungicide preparation. These play a key role in sustainable agriculture by effectively inhibiting pathogens in plants, soil, etc. They help to increase crop productivity, while their ecological nature distinguishes these Biofungicides from synthetic fungicides by supporting ecological agricultural practices and without negative impact on the population (toxicity in humans).

Advantages of the fungicidal solution of the biological preparation:

• increased effectiveness and reduction of the amount of product needed during treatment • pre- and post-infection fungicide - pre-harvest for plants and post-harvest for the so-called fumigation • sustainable release of biological compounds • protection of environmental factors • smaller losses of fungicide due to leaching, volatilization, flight and degradation in the soil • reduce phytotoxicity (damage to plants by soil fungicides - their residues) • safer application of the product reduces toxicity in humans • increases duration of action and the effectiveness of the active ingredient in the soil and at leaf level

1. Use of the preparation:

Natural fungicides are agents designed to kill pathogens in plants and in the soil, with the most common causes of disease being fungi/fungi, followed by viruses, viroids, phytoplasmas and bacteria. Natural fungicides and their use can be encountered in various areas of human activity, but most often in the growing agricultural sector, as well as organic farming (eco-farms, orchards, gardens, greenhouses, etc.) but also in urban maintenance of greenery, in horticulture, in large-capacity greenhouses etc.

2. Preparation for - organic farming:

This ecological Bio-fUngicide is suitable for use in organic farming, e.g. in the organic cultivation of hops and vines, e.g. for organic farms, gardens, etc.

Suitable for companies that use environmentally friendly chemicals, especially those that are certified according to ISO 14001:2016 (this is a globally recognized standard for EMS - environmental management systems).

- 27 CZ 38194 U1

3. The preparation is also intended for extensive large-capacity greenhouses:

For the year-round greenhouse cultivation industry - production greenhouses for the year-round cultivation of fruits, vegetables and flowers, on the scale of several hectares.

We also apply biofungicides preventively to growth media in greenhouses (during irrigation) or as a treatment to control root and crown diseases.

4. Application to the soil - treatment of the soil medium:

The most effective use of biofungicides is as a preventive soil treatment, possibly in growing substrates. They should be mixed into the growing medium before planting or applied as a drench immediately after transplanting.

5. Post-harvest applications on crops, seeds/grains, in the form of so-called fumigation:

Soil pathogens cause economic and yield losses not only in the field but even after harvest and during the storage period. In this case, it is a post-harvest treatment of plants, before storage - of crops, seeds/grains, fruits, vegetables, etc.

- wet way - the crops are soaked for a short time in the fumigation solution

- possibly with steam - seeds (treatment of grains/seeds) by causing the active substance on the plant to evaporate and take effect

Due to its antifungal and antibacterial properties, acetic acid is commonly used in the post-harvest treatment of crops - i.e. e.g. treatment of fruits, vegetables or seeds before storage, to reduce rotting and especially to extend their shelf life and shelf life.

The combination of hot water treatment with the product improves the effectiveness of fumigation and the versatility of this process. Various methods of application have been proposed to control seed-borne pathogens, e.g. steam fumigation mixing the seeds with a specific amount of the given product (in solution) or soaking the seeds in a fumigation solution, which leads to a significant increase in the number of mold-free seeds/crops and a decrease in seed germination.

Technical information for pre/post infectious fungicidal biological product users:

• Contact (the spray product forms a protective film for a certain period of time), broad-spectrum product with inhibition of mycelium growth and preventive and curative effect against fungal and other pathogens, with virucidal and bactericidal effects. After application, the causative agents of the disease (pathogenic mycelium) are disrupted and spore germination is prevented.

• This ecological Bio-fungicide is suitable for use in organic farming (spraying crops, soil and fumigation of crops) or in the organic cultivation of hops, vines and other crops, as well as for eco-farms, gardens, etc.

• Suitable for companies that use environmentally friendly chemicals, especially those that are certified according to ISO 14001:2016 (this is a globally recognized standard for EMS systems - environmental management system).

• It is a product for organic farming (natural substances) according to Council Regulation (EC) no. 834/2007 and Commission Regulation (EC) no. 889/2008 on organic production and labeling of organic products.

• The product is harmless to bees, livestock and domestic animals.

• Applying the product to the soil alone (or when spraying plants), thereby reducing the population of pathogens, soil-borne diseases caused by soil-borne pathogens.

- 28 CZ 38194 U1

It is a group of microorganisms that can cause a reduction or limitation of yield in crops. Soil borne pathogens include - fungi, bacteria and viruses.

• Various methods of applying acetic acid have been proposed to control seed-borne pathogens, e.g. acetic acid vapor fumigation, mixing seeds with a specific amount of acid or soaking seeds in acid solutions. According to research, the positive effect of acetic acid treatment persists after short-term (5 months) and long-term (12 months) storage.

• Research has confirmed that Ethanoic (acetic) acid in a concentration of 1.8% completely inhibited the growth of the mycelium of all fungi (it is a cluster of interwoven fibers, characteristic especially of fungi and some bacteria). The effectiveness of this organic compound is variable and has shown different effects on fungi based on their resistance. E.g. resistance to B. cinerea, S. sclerotiorum and F. oxysporum was higher than that of R. solani and M. phaseolina.

• GRAS compounds (so-called safe compounds - Acetic acid) were tested on Macrophomina phaseolina, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum and Rhizoctonia solani, in order to understand the effectiveness of organic acids on the development of plant pathogens.

• In general, before and after storage, regardless of temperature, the seeds of samples treated with acetic acid were characterized by a lower infestation of A. Alternata and A. Radicina than untreated seeds. In addition, acetic acid often controlled these fungi more effectively than other fungicides.

• In general, especially after storage, increasing the concentration of acetic acid (by fumigation) resulted in a significant increase in the number of mold-free seeds and a decrease in seed germination.

• Acetic acid is a natural food additive with proven antimicrobial effects.

• Research has confirmed the effectiveness of acetic acid against powdery mildew (Sphaerotheca pannosa var. Rosae, Podosphaera pannosa). Furthermore, it was found that Acetic Acid applied at 5 and 10% concentration provided good control of plant diseases, but the concentration of 0.25 and 0.5% Acetic Acid was phytotoxic.

• Acetic acid (AA) as a vapor in low concentrations was effective in preventing fruit decay by postharvest fungi. Fumigation with 2.7 or 5.4 mg AA/liter in air at 2 and 20 °C reduced germination of Botrytis cinerea Pers. and Penicillium expansum.

• Acetic acid was an effective post-harvest fumigant to kill mold spores on peaches, nectarines, apricots and cherries. The decay of Monilinia fructicola and Rhizopus stolonifer on Harbrite peaches was prevented only by 1.4 and 2.7 mg/l acetic acid. Harbrite peaches fumigated with 2.7 mg/L acetic acid were slightly injured, phytotoxicity indicated by light brown streaks.

• Citric acid (CA - citric acid) is also used for fumigation - at the same time as a disinfectant for fruit, vegetables and surfaces that come into contact with food. At the same time, this substance acts as a virucide and germicide - a chemical substance or its solution with an antimicrobial effect, which causes the inactivation or killing of the microorganism.

• Application of CA - Citric acids also induces (triggers) antioxidant defense systems, promotes increased chlorophyll content and affects secondary metabolism to limit plant growth under stress. In particular, CA has a great effect on alleviating heavy metal stress in plants

- 29 CZ 38194 U1 by promoting precipitation, chelation (binding) and sequestration (separation from the whole) of metal ions.

• Application Citric acid is quickly absorbed by the plant. Once inside, it works its way to the top of the plant. This action allows new tissue to grow without infection. Additionally, as it moves through the plant, it stimulates the production of phytoalexins (the plant's natural defense against microbial attack) that inhibit future growth of fungi and bacteria.

• The ingredients we use Salicylic acid - plays a vital role in plants in the activation of plant resistance systems to diseases, including pattern-effector-triggered immunity and systemically acquired resistance (the so-called phytohormone SA inhibits FoTOR complex 1 (FoTORCl) by activating FoSNFl in vivo). Transgenic potato plants with FoTOR1 and FoSAH1 gene interference inhibited invasive hyphal growth and significantly prevented the occurrence of Fusarium wilt. In addition, previous studies have shown that SA enhances antifungal activity by inhibiting hyphal growth and spore germination in Fusarium oxysporum, Magnaporthe grisea, and Penicillium expansum.

• Pharmacological tests have shown that exogenous (external) application of Salicylic acid can effectively stop hyphal growth, spore production and pathogenicity of F. oxysporum, while endogenous (internal) SA salicylate hydroxylases significantly detoxify the plant. Synergistic inhibition of F. Oxysporum growth was observed when SA was combined with rapamycin. Kinase assays showed that SA inhibits FoTOR complex 1 (FoTORCl) by activating FoSNF1 in vivo.

• In recent years, the area of increased interest in L-ascorbic acid is also pest control. Vitamin C, together with salicylic acid, has been found to be a key component of the so-called oxygen burst, which is a necessary part of the system - Activated resistance (SAR) and induced - Systemic resistance (ISR) in plants.

• Studies suggest that vitamin C (L-ascorbic acid) can show significant fungicidal activity against Fusarium graminearum, Alternaria alternata, F. solani, Macrophomina phaseolina, and Pyrenophora tritici-repentis in vitro, under certain conditions also against Magnaporthiopsis maydis, which causes the disease late wilt in maize. Other research confirms that L-ascorbic acid can reduce linear growth on plant pathogens such as Fusarium oxysporum, F. solani and Macrophomina phaseolina.

• L-ascorbic acid (vitamin C) - has strong antimicrobial properties that reduce the pathogenicity of bacteria, viruses, parasites and fungi.

• In 2013, the European Commission approved vitamin C as an effective substance for the treatment of potatoes and tomatoes against plant pathogens such as: Fusarium, Phytophthora infestans and Botrytis sp. Treatment of foliar diseases of vegetables with Vit. C, was able to significantly reduce powdery mildew infestation.

• Vitamin C is recognized as effective in fighting fungal/fungal and viral infections. It helps protect the plant from water stress, ozone and UV radiation. Used in the process of photosynthesis, it can regulate cell growth. It also serves as a redox buffer - it is a pair of substances that change into each other through the gain/loss of one proton.

• L-ascorbic acid (Vit. C) regulates abiotic and biotic stress reactions, thereby significantly reducing the growth of bacterial or fungal pathogens.

• L-ascorbic acid has its role in the regulation of photosynthesis and also as an antioxidant detoxifying exogenous and endogenously generated ROS.

• Conducted research has demonstrated the strong antifungal activity of L-ascorbic acid against

- 30 CZ 38194 U1 mycelia and conidiospores of Alternaria solani and Venturia inaequalis • The role of L-ascorbic acid in defense against pathogens has received only limited attention. In studies to date, the content of L-ascorbic acid led to a reduction in the growth of the bacterial pathogen when attacked by pathogens - Arabidopsis vtc1 (mutant) or vtc2, as well as in Pseudomonas syringa pv maculicola and inhibition of hyphal growth of the pathogen Peronospora parasitica.

• The substance with fungicidal and antibacterial effects is the so-called L-ascorbic acid. phytovaccine, as numerous studies have shown that it has a wide range of antimicrobial activities and acts as a botanical fungicide, inhibiting spore germination and growth of plant pathogens.

• Antifungal in vitro tests with one of the most popular medicinal substances in the world: L - ascorbic acid, widely known as vitamin C, were carried out with the three most widespread plant pathogens: Monilia fructigena, Alternaria solani and Venturia inaequalis in order to evaluate the direct antifungal action of the acid used in agriculture as an ISR (phytovaccine) promoter. Thus, vitamin C has strong antimicrobial properties reducing the pathogenicity of bacteria, viruses, parasites and fungi, which means that vitamin C can be used as a direct fungicide and phytovaccine in pest control. In addition, the treatment reduced the occurrence of several genera of fungi, i.e. Alternaria, Aspergillus, Fusarium, Penicillium, Rhizoctonia, Rhizopus, Stemphylium, Trichothecium and Verticillium.

• The preparation also works on the overwintering stages of pests, as a preventive spray in the spring (application February, March).

Instructions for use of fungicide - indications, protection against diseases:

The use is suitable as a preventive and at the same time as a curative, especially on - vines and hops, (flowers and heads are more sensitive than leaves), agricultural crops, also - fruits, vegetables, useful and ornamental plants, etc.

Preventive effect by spraying - protects the plant from the appearance of the disease, it is necessary to apply before the first manifestation of the disease, at the same time the product also protects the soil in order to reduce the population of pathogens.

The preventive use of biofungicides is - treatment also in growing substrates (material for growing plants/soil spraying), or as treatment of crops and seeds after harvest in the form of fumigation. It also acts on overwintering stages of pests, as a preventive spray in spring (application February, March).

We also apply biofungicides preventively to growing media (soil irrigation) or as treatments to control root and crown diseases and can be just as effective as chemical fungicides. Biofungicides used to treat foliar diseases must be applied preventively, as they are safer for the grower, can be more persistent (resistant), and can sometimes be cheaper than conventional synthetic fungicides.

Curative (healing) effect by spraying - i.e. it protects the plant after being attacked by pathogens, i.e. even after infection, it treats plants affected by the disease (even if the disease is not yet visible to the eye), at the same time it treats a plant with a viral disease transmitted by sucking insects.

Dilution of the concentrate with water: spraying according to the occurrence of infestation and according to the preventive effect (the spray mist should contact all parts of the plant), including the so-called crop and irrigation medium fumigation:

Mix/shake well before use and after diluting the concentrate.

1) dilution of the product 1:12 / application by spraying - before flowering (13 l is enough for 150 m ² ) from 13 to

- 31 CZ 38194 U1 °C - before flowering and preventively after flowering - at moderate pressure after plants are hit by pathogens

2) dilution of the preparation 1:8 / application by spraying - after flowering (9 l is enough for 100 m ² ) from 13 to 27 °C - strong impact of pathogens on plants

3) dilution of the preparation 1:12 / post-harvest fumigation/disinsection of crops and grains/seeds (with solution, possibly steam for seeds/grains, it is a post-harvest application on crops, seeds, etc.) from 16 to 25 °C

4) dilution of the product 1:8 / treatment of soil, soil pathogens February - March, by spraying or in greenhouses in the form of an irrigation medium / during watering, continuously (9 l is enough for 100 m2)

It is generally recommended to spray one or two times before flowering (hops, vines, etc.). 2-3 treatments after flowering are very important:

• Vine - the most important treatment is during the ripening of the vine berries, when the vine is most susceptible to attack. Hops and Vines according to the table and BBCH (otherwise according to the signalling) • spray on all parts of the plant (leaves, stems, crowns, etc.), a light spray falling on the surface of the soil can help reduce unwanted yeast and mold in the soil itself • natural bio- fungicide is most effective in warmer weather when weeds are drier. Plan the use of the spray well, it should not rain for at least 3 hours after application, you should not even water to make the effect as effective as possible.

• growing crops after application - protection period/OL (approx. 24 hours) of the product - then without restrictions

Frequency: Apply when conditions are favorable for disease development. Apply at 8 to 16 day intervals depending on disease pressure and environmental conditions (also according to BBCH and signaling).

Preparation of fungicide: dilution of concentrate for other crops - application by spraying during vegetation and post-harvest application on stored crops by fumigation.

To achieve better results, it is advisable to spray directly on the plants, in sunny weather.

Timing: Apply diluted spray after irrigation, NOT before rain. Do not water the treated areas for approximately 12 hours to ensure that the product is not washed off the surface of the leaves.

OL 1 (withdrawal period 1 day) of crops - fruits, vegetables, herbs, etc., is - 1 day (withdrawal period - is the time in days that must be observed from the last application of the preparation to the beginning of the harvest of the given crop, most often because the protection period is determined by the date of application, for example before flowering or after planting, the so-called BBCH).

Use of the pre/post infectious Bio-fungicide preparation in the relevant period:

- in the growing season April to October

- for overwintering stages of pests, as preventive soil spraying in spring (application February, March)

- in large-capacity greenhouses - continuously

- OL 1 - protection period 1 day

The product is harmless to bees, livestock and domestic animals (after drying). This product can be used in organic farming according to Council Regulation (EC) no. 834/2007 and Commission Regulation (EC) no. 889/2008 on organic production and labeling of organic products

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The table intended for the treatment of hops and grapevines: until BBCH 61 - before flowering, from BBCH 61 - after flowering (other crops are listed below - spraying according to the occurrence of infestation and according to the preventive effect).

Crop, area of use Harmful organism. Dosage, missionability OL A note on the crop Interval between applications Vine BBCH indicates an internationally used scale of fruiting and growth stages of plants Downy mildew (Peronospora) to BBCH 61 1:8 5 1 per 100 m ² from BBCH61 1:12 6 1 per 100 m ² 1 Preventive (during the flowering of the VINE, it is not recommended to chemically interfere with the plants in any way and thus increase the risk of poor flowering (apply before flowering and after flowering, according to BBCH 8 - 16 days (otherwise as needed We provide protection against mold in particular in the form of 4-5 protective interventions in the period from approximately mid-June to mid-August. Hops Hop mold (Pseudoperonospora humuli) to BBCH 61 1:8 5 1 per 100 m ² pdBBCHól 1:12 1 As a preventive measure, according to the signaling (during the flowering of HOPS, it is not recommended to chemically interfere with the plants in any way and thus increase the risk of poor flowering (apply before flowering and after flowering, according to BBCH 8-16 days (otherwise as needed

Shelf life of the product (expiration): 1 year from the date of manufacture with the correct storage method:

- store in original, closed, intact packaging

- store in dark warehouses, at a temperature of +8 to +20 °C.

- store separately from food, feed, chemicals, flammables, medicines, disinfectants

Keep out of reach of children. The product may cause eye irritation. Repeated contact may cause skin irritation. Inhalation of vapor may cause respiratory irritation.

You can apply the product to plants/crops:

- can be used in all phases of plant growth, even for viral diseases transmitted by sucking insects

- green vegetables, veneer vegetables, fruit trees, ornamental plants, lawns, herbs

- e.g. peppers, tomatoes, cucumbers, gourds (zucchini), vine cores and vine seeds, fruit bushes, etc.

- for use on field crops - vines, hops, ornamentals, lawns, fruit orchards, citrus, vegetables, flowers and other crops.

Can also be applied as a root dip, band spray, seed treatment, spot application or surface spray.

Post-harvest applications to crops before storage:

application of the product in the form of post-harvest fumigation with a solution - crop disinsection / steam fumigation - cereal grains, etc. Combining hot water treatment with the product improves the efficiency and versatility of this process. It is fumigation in steam, event, mixing of seeds/crops with a specific amount of preparation in the form of soaking seeds/crops in solutions.

Spraying of soil material in the growing season:

this is one of the most effective uses of biofungicides as a preventive treatment in growing substrates (material for growing plants). The fungicide should be mixed into the growing medium before planting or applied as an irrigation immediately after transplanting.

Spraying the soil mass before the growing season - February, March:

effective also on overwintering stages of pests, application by spraying - February, March (e.g. against Sviluška - which causes blisters by sucking on the leaves, the leaves turn yellow and turn gray, with strong occurrences they dry up and fall off).

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Technical overview of the plant diseases we deal with:

This product controls diseases, especially fungi, as well as viruses, viroids, phytoplasmas and bacteria.

• Fusarium - Fusarium wilt is a fungal plant disease caused by fungi of the genus Fusarium from the Nectriaceae family. Probably the most common pathogen is Fusarium oxysporum. The same genus causes Fusarium rot of apples and attacks potatoes or corn.

• Gray mold - Botrytis cinerea • Snow mold - Monographella nivalis (lawns) • Powdery mildew - Blumeria, Erysiphe • Vine powdery mildew - Uncinula necator • Vine mold - Peronospora vyve, Plasmopara viticola • Hop mold - Pseudoperonospora humuli • Fruit rot, moniliosis - Monilinia fructigena (apples, pears, etc.) • Originators rust - Melampsora, Puccinia, Uromyces, a group of parasitic stem-spore fungi, these are parasites of plants that form a subfungus or mycelium • Bacterial leaf spot - Stigmima carpophila, Diplocarpon mespili, etc.

• Root and stem rot - Sclerotinia sclerotiorum • Bacterial rot, bacterial wilt, bacterial leaf spot

Industrial applicability

It is a biological fungicide product with three uses (pre- and post-infection fungicide - for plants, then for soil treatment with fungicide and post-harvest product for the so-called fumigation of crops/grains):

- for the treatment of plant fungi, viruses - Fungicide/Bactericide/Virucide (fungicidal use on plants) - for the treatment of soil pathogens (spraying the soil, e.g. before sowing, or as an irrigation medium especially in greenhouses)

- for fungicidal post-harvest applications on stored crops (wet fumigation - crops, steam fumigation - seeds/grains)

The use of bio-based natural fungicides is less hazardous to human health and the food base (without synthetic chemicals), while the potential of improved bio-formulation systems helps address current crop treatment issues and presents the benefits of bio-fungicides that are not harmful to the wider environment.

Claims (2)

PROTECTION CLAIMS 1. Fungicidal agent, characterized in that it contains citric acid in an amount from 0.06% to 39% by weight. agent, salicylic acid in amounts from 0.08% to 28% 5 wt. preparation, L-ascorbic acid in an amount from 0.06% to 36% by weight. agent, glycerin in amounts from 0.3% to 42% by weight. of the resource and the balance up to 100% wt. this fungicide is made up of acetic acid. 2. The fungicide according to claim 1, characterized in that it is in the form of a solution.